Viability and maturation of Aphanomyces cochlioides oospores

Plasmolysis, tetrazolium bromide staining and microscopic appearance were tested for their usefulness in determining viability of oospores of Aphanomyces cochlioides. For comparison, three lethal treatments were employed to contrast the reaction of dead oospores and untreated, presumably viable oospores. Few oospores stained with tetrazolium bromide, even though plasmolysis and microscopic appearance indicated that 85% were viable. Cytoplasm of viable oospores was densely organized and uniformly granular (DOUG), whereas cytoplasm of oospores exposed to lethal treatments was loosely organized and non-uniformly granular (LONG). Dose-response bioassay experiments were conducted with untreated oospores of varying inoculum densities or with mixtures of untreated DOUG and heat-treated LONG oospores in varying proportions. The number of DOUG oospores was correlated (R(2) = 0.62, P < 0.001) with severity of damping-off of sugar beet seedlings caused by A. cochlioides. Thus, the granular appearance of cytoplasm offered a fast, easy and reliable indicator of viability of A. cochlioides oospores. Tests with newly formed oospores/oogonia showed that >80% harvested at 3-4 d after inoculation of hypocotyls stained with tetrazolium, but by 8-9 d <10% stained, apparently because of declining permeability of the spore wall to tetrazolium as oospores matured.     

Nicotinamide and structurally related compounds show halting activity against zoospores of the phytopathogenic fungus Aphanomyces cochlioides

In a survey of plant secondary metabolites regulating the behavior of phytopathogenic Aphanomyces cochlioides zoospores, we found that leaf extracts of Amaranthus gangeticus and cotyledon extracts of pea (Pisum sativum) remarkably halted the motility of zoospores. Bioassay-directed fractionation of A. gangeticus and pea constituents revealed that the halting activity was dependent on a single chemical factor (halting factor). The active principle was identified as nicotinamide (1) by comparing its biological activity and spectroscopic properties with those of the authentic compound. Nicotinamide (1) showed potent halting activity toward the zoospores of A. cochlioides and A. euteiches, but it exhibited very less activity against other Oomycetes, Pythium aphanidermatum and Phytophthora infestans zoospores. Interestingly, the zoospores halted by nicotinamide (1) encysted within 10-15 min and then the resulting cystospores regenerated zoospores instead of germination. Nicotinamide (1) and related compounds were subjected to the halting activity bioassay to elucidate the structure-activity relationships. These bioassays revealed that part structures of (A) the aromatic ring containing at least one nitrogen atom, (B) carbonyl-like group adjacent to the aromatic ring and (C) hydrogen atoms on the amide group are responsible for the strong activity. So far, this is the first report of halting activity of nicotinamide (1) against fungal zoospores.     

Experiments with fungicides to control Aphanomyces cochlioides in sugar beet

Aphanomyces cochlioides attacking sugar-beet seedlings was controlled by fenaminosulf applied as seed treatment or to the seed furrow at sowing. In the absence of blackleg caused by A. cochlioides, fenaminosulf seed treatment at 0–75 % (of seed weight) and soil applications decreased seedling emergence, and soil applications of more than 2-2 kg a.i./ha significantly depressed sugar yield. Controlling moderate attacks of seedling blackleg caused by A. cochlioides did not increase yield. Dichlone, benomyl and maneb applied as seed or soil treatments, captafol seed treatment, and quintozene and thiram soil treatments did not control A. cochlioides.     

Chemotaxis of Fungal Zoospores,with Special Reference to Aphanomyces cochlioides

Zoospores of phytopathogenic fungi accumulate at the potential infection sites of host roots by chemotaxis. The aggregated spores then adhere, encyst, germinate, and finally penetrate into the root tissues to initiate infection. Some of the host-specific attractants have already been identified. The host-specific attractants also induce cell differentiation of certain zoospores under laboratory conditions. This indicates that a signal released from the roots of the host plant guides the pest propagules for orientation and prepares them for establishing a host-pathogen relationship by necessary physiological changes. Some non-host plant secondary metabolites were found to markedly regulate behavior and viability of zoospores, suggesting that non-host compounds may also play a role in protecting the non-host plants from the attack of zoosporic fungi. We hypothesized that zoospores perceive the host signal(s) by specific G-protein-coupled receptors and translate it into responses by way of the phosphoinositide-Ca²⁺ signaling cascade. The details of the signal transduction mechanism in fungal zoospores are yet to be discovered. In this report, we review the signaling and communications between phytopathogenic fungal zoospores and host and non-host plants with special reference to Aphanomyces cochlioides.     

Chemotaxis of fungal zoospores, with special reference to Aphanomyces cochlioides.

Zoospores of phytopathogenic fungi accumulate at the potential infection sites of host roots by chemotaxis. The aggregated spores then adhere, encyst, germinate, and finally penetrate into the root tissues to initiate infection. Some of the host-specific attractants have already been identified. The host-specific attractants also induce cell differentiation of certain zoospores under laboratory conditions. This indicates that a signal released from the roots of the host plant guides the pest propagules for orientation and prepares them for establishing a host-pathogen relationship by necessary physiological changes. Some non-host plant secondary metabolites were found to markedly regulate behavior and viability of zoospores, suggesting that non-host compounds may also play a role in protecting the non-host plants from the attack of zoosporic fungi. We hypothesized that zoospores perceive the host signal(s) by specific G-protein-coupled receptors and translate it into responses by way of the phosphoinositide-Ca2+ signaling cascade. The details of the signal transduction mechanism in fungal zoospores are yet to be discovered. In this report, we review the signaling and communications between phytopathogenic fungal zoospores and host and non-host plants with special reference to Aphanomyces cochlioides.     

Repellent activity of estrogenic compounds toward zoospores of the phytopathogenic fungus Aphanomyces cochlioides

Screening chemical compounds, we found that a xenoestrogen, bisphenol A, showed potent repellent activity against the zoospores of Aphanomyces cochlioides. Based on this finding, we tested a number of androgenic and estrogenic compounds (e.g. testosterone, progesterone, estradiols, diethylstilbestrol, estrone, estriol, pregnenolone, dienestrol etc.) on the motility behavior of A. cochlioides zoospores. Interestingly, most of the estrogenic compounds exhibited potent repellent activity (1 microg/ml or less by the "particle method") toward the motile zoospores of A. cochlioides. We derivatized some of the estrogens and discussed the relationship between the structure of active molecules and their repellent activity. Apparently, aromatization of the A ring with a free hydroxyl group at C-3 position of a steroidal structure is necessary for higher repellent activity. Interestingly, methylation of diethylstilbestrol (DES) yielded completely different activity i.e. both mono- and di-methyl ethers of DES showed attractant activity. Moreover, the attracted zoospores were encysted and then germinated in the presence of di-methyl ether of DES. The potential usefulness of this repellent test is discussed for the detection of estrogenic activity of naturally occurring compounds, and the possible role of phytoestrogens in host/parasite interactions. So far, this will be the first report of repellent activity of estrogenic compounds toward trivial fungal zoospores.     

Synergistic effects of anacardic acids and methicillin against methicillin resistant Staphylococcus aureus

The synergistic effects of 6-alk(en)ylsalcylic acids, also known as anacardic acids, in combination with methicillin against Staphylococcus aureus ATCC 33591 (MRSA) was investigated. The double bond in C15-anacardic acids is not essential in eliciting the antibacterial activity but is associated with increasing the activity. The synergistic effects decreased with increasing the number of double bonds in the alkyl chain. On the other hand, the antibacterial activity of anacardic acids possessing different alkyl chain lengths against the same MRSA strain was found to be a parabolic function of their lipophilicity and maximized with the alkyl chain length of C10 and C12. Notably, the synergistic effects were noted to increase with increasing the alkyl chain length.     

Secondary metabolites from nonhost plants affect the motility and viability of phytopathogenic Aphanomyces cochlioides zoospores

The motile zoospores of the damping-off pathogen Aphanomyces cochlioides aggregate on host plants (e.g., sugar beet, spinach) guided by the host-specific plant signal cochliophilin A before infection. To assess the potential role of secondary metabolites in nonhost resistance, acetone extracts of 200 nonhost traditional medicinal plants from Chinese and Bangladeshi origins were tested for the motility behaviour of A. cochlioides zoospores using a particle bioassay method. Nearly one third of the tested plant extracts exhibited diverse deleterious activities such as repellent, stimulant, motility halting and lysis against A. cochlioides zoospores. Among these active plants, an extract of the Chinese medicinal plant Dalbergia odorifera displayed potent repellent activity toward zoospores. Chromatographic separation of D. odorifera constituents revealed that the repellent activity was regulated by the cumulative effect of three motility-affecting isoflavonoids, viz. (+/-)-medicarpin (repellent at 150 microg/ml), (-)-claussequinone (stimulant at 100 microg/ml) and formononetin (stimulant and attractant at 50 microg/ml). A mixture (1:1:1, w/w/w) of these three compounds exhibited only repellent activity toward zoospores at a concentration lower than 50 microg/ml. These results suggest that nonhost plants might possess potential bioactive secondary metabolites to ward off zoosporic phytopathogens.     

Phenolic constituents of Celosia cristata L. susceptible to spinach root rot pathogen Aphanomyces cochlioides

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), known as a host-specific attractant towards the zoospores of Aphanomyces cochlioides, a cause of root rot and damping-off diseases of Chenopodiaceae, was found in the Amaranthaceae plant, Celosia cristata, that is susceptible to the pathogen. The content of 1 in Celosia seedlings was quantified as 1.4 microg/g fresh weight. A new isoflavone, cristatein (5-hydroxy-6-hydroxymethyl-7,2'-dimethoxyisoflavone, 2), and five known flavonoids were also identified.     

Quantitative trait locus responsible for resistance to Aphanomyces root rot (black root) caused by Aphanomyces cochlioides Drechs. in sugar beet

Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet (Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants’ phenotypic and molecular markers’ segregation was carried out. At a highly infested field site, some 187 F₂ and 66 F₃ individuals, derived from a cross between lines ‘NK-310mm-O’ (highly resistant) and ‘NK-184mm-O’ (susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 ‘NK-310mm-O’-coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F₂ population (in the year 2002) and 65% in F₃ lines (2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.     

Evaluation of lipoxygenase inhibitory activity of anacardic acids

6-Alkylsalicylic acids inhibit the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) competitively and without pro-oxidant effects. This activity is largely dependent on the nature of their alkyl side chains. Inhibitory activities of anacardic acids, viz. 6-pentadec(en)ylsalicylic acids, isolated from the cashew Anacardium occidentale, were initially used for comparison because their aromatic head portions are the same. Consequently, the data should be interpreted to mean that changes in the hydrophobic side chain tail portions of the molecules evaluated correlate with the specific activity determined.     

A photoaffinity probe designed for host-specific signal flavonoid receptors in phytopathogenic Peronosporomycete zoospores of Aphanomyces cochlioides

Aphanomyces cochlioides zoospores show chemotaxis to cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), a host derived attractant, and also respond to 5,7-dihydroxyflavone (2) known as an equivalent chemoattractant. To investigate the chemotactic receptors in the zoospores, we designed photoaffinity probes 4'-azido-5,7-dihydroxyflavone (3) and 4'-azido-7-O-biotinyl-5-hydroxyflavone (4) considering chemical structure of 2. Both 3 and 4 had zoospore attractant activity which was competitive with that of 1. When zoospores were treated with the biotinylated photoaffinity probe followed by UV irradiation and streptavidin-gold or peroxidase-conjugated streptavidin, probe-labeled proteins were detected on the cell membrane. This result indicated that the 1-specific-binding proteins, a candidate for hypothetical cochliophilin A receptor, were localized on the cell membrane of the zoospores. This is the first experimental evidence of flavonoid-binding proteins being present in zoospores, using chemically synthesized azidoflavone as photoaffinity-labeling reagent.     

Characterization of xanthine oxidase inhibition by anacardic acids

Anacardic acid, 6[8(Z), 11(Z), 14-pentadecatrienyl]salicylic acid, inhibits generation of superoxide radicals by xanthine oxidase. This inhibition does not follow a hyperbolic inhibition, depends on anacardic acid concentrations, but follows a sigmoidal inhibition. The inhibition was analyzed by using a Hill equation, and slope factor and EC(50) were 4.3+/-0.5 and 53.6+/-5.1 microM, respectively. In addition, anacardic acid inhibited uric acid formation by xanthine oxidase cooperatively. Slope factor and EC(50) were 1.7+/-0.5 and 162+/-10 microM, respectively. The results indicate that anacardic acid binds to allosteric sites near the xanthine-binding domain in xanthine oxidase. Salicylic acid moiety and alkenyl side chain in anacardic acid are associated with the cooperative inhibition and hydrophobic binding, respectively.     

The third naturally occurring attractant toward zoospores of phytopathogenic Aphanomyces cochlioides from the Spinacia oleracea host plant

A bioassay-guided survey of spinach leaf constituents resulted in 5,4'-dihydroxy-3,3'-dimethoxy-6,7-methylenedioxyflavone being identified as the third naturally-occurring attractant in the host plant toward the zoospores of its pathogen, Aphanomyces cochlioides. The isolate showed attracting activity around Chromosorb W AW particles (60-80 mesh) coated with a 10(-5) M solution in a zoospore suspension. However, this activity was 1/100-1/1000 less than that of cochliophilin A, an attractant in the roots of spinach. Bioassays with the present isolate and related compounds revealed that 5,3',4'-trihydroxy-3-methoxy-6,7-methylenedioxyflavone did not possess attractant activity, but rather weak antagonistic activity toward the former two attractants from spinach.     

Dynamic rearrangement of F-actin organization triggered by host-specific plant signal is linked to morphogenesis of Aphanomyces cochlioides zoospores

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), a root releasing host-specific plant signal triggers chemotaxis and subsequent morphological changes in pathogenic Aphanomyces cochlioides zoospores before host penetration. The present study illustrates time-course changing patterns of cytoskeletal filamentous actin (F-actin) organization in the zoospores of A. cochlioides during rapid morphological changes (encystment and germination) after exposure to cochliophilin A. Confocal laser scanning microscopic analysis revealed that F-actin microfilaments remained concentrated at ventral groove and diffusely distributed in peripheral cytoplasm of the zoospore. These microfilaments dramatically rearranged and changed into granular F-actin plaques interconnected with fine arrays during encystment. A large patch of actin arrays accumulated at one pole of the cystospores just before germination. Then the actin plaques moved to the emerging germ tube where a distinct cap of microfilaments was seen at the tip of the emerging hypha. Zoospores treated with an inhibitor of F-actin polymerization, latrunculin B or motility halting and regeneration inducing compound nicotinamide, displayed different patterns of F-actin in both zoospores and cystospores than those obtained by the induction of cochliophilin A. Collectively, these results indicate that the host-specific plant signal cochliophilin A triggers a dynamic polymerization/depolymerization of F-actin in pathogenic A. cochlioides zoospores during early events of plant-peronosporomycete interactions.     

Mode of antagonism of a biocontrol bacterium Lysobacter sp. SB-K88 toward a damping-off pathogen Aphanomyces cochlioides

The biocontrol bacterium Lysobacter sp. SB-K88 suppresses damping-off disease in sugar beet and spinach caused by Aphanomyces cochlioides and Pythium sp. through characteristic plant colonization and antibiosis against the pathogens. This study aimed to unravel further details on mode of antagonism of SB-K88 against a damping-off pathogen A. cochlioides AC-5. The SB-K88 substantially inhibited growth and decomposed AC-5 mycelia and suppressed the release of zoospores from the hyphae. The excised root tips of sugar beet seedlings from seeds previously inoculated with SB-K88 were less attractive to AC-5 zoospores. Although aerial growth was not affected, however, root hairs of SB-K88 inoculated sugar beet seedlings were remarkably shorter and thicker than those of uninoculated control. When exposed to zoospores, the SB-K88 inhibited motility of zoospores and/or caused lysis, and then aggregated around the dead cystospores or lysed residues within 3–6 h likely to be micro-predatory behavior to a eukaryotic organism. Confocal laser scanning microscopic analysis revealed that number of lipid bodies and activities of mitochondria were markedly increased in the affected hyphae compared with control hyphae as visualized by established vital stains. Taken together, these results suggest that Lysobacter sp. SB-K88 suppresses damping-off diseases through exerting multifaceted antagonistic effects against the peronosporomycetes.     

Synthesis of anacardic acids in seeds of Ginkgo biloba.

Anacardic (6-alkylsalicylic) acids and common lipids are efficiently synthesized by immature seeds of Ginkgo biloba. The seeds were incubated with 14C-labeled acetic, malonic and palmitoleic acids, glucose, and other potential precursors. Levels of 14C in common lipids and in anacardic acids, and the distribution of 14C in anacardic acids were determined. The results show that the salicylic moiety is synthesized by a polyketide pathway via malonic acid. The chain moiety for anacardic acid synthesis is in a different state of activation and/or site than chains that are used for synthesis of the common lipids. Labeled shikimic acid did not contribute 14C to anacardic acids, nor to other lipids, and palmitoleic acid was incorporated only into common lipids.     

Host-specific plant signal and G-protein activator, mastoparan, trigger differentiation of zoospores of the phytopathogenic oomycete Aphanomyces cochlioides

We found that the gradient of a host-specific attractant, cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone) isolated from the roots of spinach triggered encystment followed by germination of zoospores of Aphanomyces cochlioidesat a concentration less than micromolar order. This compound did not affect the growth and reproduction of this phytopathogen up to 10^–6 M concentration in the culture medium. We also observed that mastoparan, an activator of heterotrimeric G-protein could inhibit the motility of zoospores and then strikingly effect encystment followed by 60–80% germination of cysts. Concomitant application of cochliophilin A and mastoparan showed stronger encystment followed by 100% germination of cysts. In addition, we have observed that chemicals interfering with phospholipase C activity (neomycin) and Ca²⁺ influx/release (EGTA and loperamide) suppress cochliophilin A or mastoparan induced encystment and germination. These results suggest that G-protein mediated signal transduction mechanism may be involved in the differentiation of the A. cochlioides zoospores. This is the first report on the differentiation of oomycete zoospores initiated by a host-specific plant signal or a G-protein activator.     

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.